Note: Descriptions are shown in the official language in which they were submitted.
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USE OF NATURAL SUBSTANCES AS FEED ADDITIVES FOR AQUATIC
ANIMALS
The present invention relates to the use of natural active substances selected
from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, di-
hydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of feed
for aquatic animals including fish and shrimp, especially for cold water fish
as for
example salmon, bream, bass and for warm water fish as for example carp,
tilapia,
catfish.
More particular, this invention relates to the use of at least two substances
as de-
fined above for the improvement of the feed conversion ratio and/or daily
weight
gain in fish, for reducing mortality by regulating the micro flora of the gut
and/or by
protecting the animal against infections caused by pathogenic microorganisms.
Furthermore, the present invention relates to a novel fish feed composition
com-
prising as active ingredients at least two, preferably at least three or four
active
compound(s) selected from the group consisting of alpha-pinene, alpha-
terpineol,
cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene.
The term feed or feed composition means any compound, preparation, mixture, or
composition suitable for, or intended for intake by an animal.
One important factor in aquaculture is the turnover rate. Turnover rate is
deter-
mined by how fast the fish grow to a harvestable size. As an example, it takes
from 12 to 18 months to raise Atlantic salmon from smolt (the physiological
stage
when the Atlantic salmon can first be transferred from fresh water to sea
water) to
harvestable size. A fast turnover has several positive results. First, it
helps cash
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flow. Second, it improves risk management. Especially, a high mortality rate
is a
substantial risk for fish farmers.
It is generally known that mortality rate increases by an unbalanced
microflora
and/or by infections caused by pathogenic microbes. Fish diseases are common,
and the likelihood of an outbreak is higher over a long growing period. There
is
also a risk that fish will escape due to accidents, e.g. when shifting nets,
or due to
bad weather causing wrecked fish pens.
For other farm animals it is well known to use antibiotics and vaccines to
prevent
the development of diseases. In aquaculture, antibiotics are not so much used -
at
least in cold water aquaculture - due to the fact that disease spread very
quickly,
diseased fish do not eat much and also due to the negative impact on the envi-
ronment of the wasted medicated feed. Vaccines are widely used when available
but they are not developed for all diseases.
As an alternative to synthetic drugs, the use of plant extracts and essential
oils in
animal feed is described in the literature. For example, patent W02004/091307
describes the use of polyphenols, and other natural actives in feed to
increase
survival rate of Artemia after hatching. W02004/091307 is however silent with
re-
gard to the selection of compounds to be used. Moreover, the application of
poly-
phenols to reduce mortality is in the disclosed case above only useful at time
of
hatching.
It therefore remains a need in aquaculture to prevent the development of dis-
eases, thereby reducing mortality by any prophylactic means including
antimicro-
bial activity at the gut level.
The inventors of the present application surprisingly found that substances as
de-
fined above have a great potential for use in fish feed, e.g. for improving
the feed
conversion ratio (FCR) and/or weight gain and/or for the modulation of the gut
flora. Further, the inventors surprisingly found that the substances, which
are
hereinafter also referred to as compounds, have also antimicrobial activity
result-
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ing in a reduced mortality. The unique selection of active compounds of the
present
invention allows for the first time controlling a number of fish diseases
caused by a
number of different pathogens.
Therefore, in a first particular embodiment, the invention relates to methods
for using
at least two active compounds selected from the group consisting of alpha-
pinene,
alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and
terpinolene in fish feed for improving the Feed Conversion Ratio (FCR) and/or
weight
gain and/or for reducing mortality by modulation of the gut micro-flora and/or
by
preventing diseases caused by pathogenic microorganisms. For example, it has
been
shown that selected compounds of the invention (eg.: cinnamaldehyde,
dihydroeugenol, eugenol, and meta-cresol exhibit excellent effects inhibiting
the
growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric
Redmouth (ERM), a disease found especially in salmonids and causing high
mortality.
In alternative embodiments, alpha-pinene and/or alpha-terpineol and/or
cinnamaldehyde and/or dihydroeugenol and/or eugenol and/or meta-cresol and/or
terpinolene is/are used to improve animal feed digestibility and/or maintain
animal
health by supporting immune system function.
In another embodiment, the invention relates to methods for using at least
three
active compounds selected from the group consisting of alpha-pinene, alpha-
terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and
terpinolene in
fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain
and/or
for reducing mortality by modulation of the gut microflora and/or by
preventing
diseases caused by pathogenic microorganisms.
In a preferred embodiment, alpha-pinene and cinnamaldehyde are used to improve
animal feed digestibility and/or maintain animal health by supporting immune
system
function.
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In another embodiment, there is provided use of alpha-pinene and
cinnamaldehyde in
a feed composition for improving feed conversion ratio and/or daily weight
gain in
aquatic animals.
In another embodiment, there is provided use of alpha-pinene and
cinnamaldehyde in
a feed composition for regulating the micro flora of the gut in aquatic
animals.
In another embodiment, there is provided use of alpha-pinene and
cinnamaldehyde in
a feed composition for reducing mortality in aquatic animals.
In another embodiment, there is provided use of alpha-pinene and
cinnamaldehyde
for treatment and prevention of diseases caused by pathogenic microorganisms
in
aquatic animals.
In another embodiment, there is provided use of a mixture of alpha-pinene and
cinnamaldehyde in a feed composition for treatment and prevention of diseases
caused by Aeromonas salmonicida in aquatic animals.
In another embodiment, there is provided use of a mixture of alpha-pinene and
cinnamaldehyde in a feed composition for protecting aquatic animals against
furunculosis caused by Aeromonas salmonicida.
In another embodiment, there is provided a feed composition or a premix
composition, or a feed additive for aquatic animals thereof, comprising alpha-
pinene
and cinnamaldehyde.
In another embodiment, there is provided a feed additive as described herein,
wherein the feed additive composition further comprises meta-cresol and
dihydro-
eugenol.
In another embodiment, there is provided a method for improving the feed
conversion
ratio in aquatic animals, wherein alpha-pinene and cinnamaldehyde are added to
the
animal feed.
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In another embodiment, there is provided a method for improving feed
conversion
ratio and/or daily weight gain in aquatic animals and/or for regulating the
micro flora
of the gut and/or for protecting the aquatic animals against infections caused
by
pathogenic microorganisms, which method comprises providing to the aquatic
animals for ingestion a feed comprising an effective amount of alpha-pinene
and
cinnamaldehyde.
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The FCR may be determined on the basis of a fish growth trial comprising a
first
treatment in which a mixture of at least two compounds according to the
invention
is added to the animal feed in a suitable concentration per kg feed, and a
second
treatment (control) with no addition of the compound(s) to the animal feed.
As it is generally known, an improved FCR is lower than the control FCR. In
particular embodiments, the FCR is improved (i.e., reduced) as compared to the
control by at least 1.0 %, preferably at least 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9
%,
2.0 %, 2.1 %, 2.2 /0, 2.3 %, 2.4 %, or at least 2.5 %.
The term "gut" as used herein designates the gastrointestinal or digestive
tract
(also referred to as the alimentary canal) and it refers to the system of
organs
within multi-cellular animals which takes in food, digests it to extract
energy and
nutrients, and expels the remaining waste.
The term gut "microflora" as used herein refers to the natural microbial
cultures
residing in the gut and maintaining health by aiding in proper digestion.
The term "modulate" as used herein in connection with the gut microflora
generally means to change, manipulate, alter, or adjust the function or status
thereof in a healthy and normally functioning animal, i.e. a non-therapeutic
use.
The term "supporting immune system function" as used herein refers to the
immune stimulation effect obtained by the compounds.
The term "mortality" as used herein refers to the ratio of life animals at the
end of
the growth phase versus the number of animals originally included into the
pond.
It may be determined on the basis of a fish challenge trial comprising two
groups
of fish challenged by a particular fish pathogen with the aim to provoke a
mortality
of 40 to 80 % of the animals in the untreated group. However, in the challenge
group fed with a suitable concentration per Kg of feed of a mixture of at
least two
compounds according to the invention, the mortality is reduced compared to the
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untreated group by at least 5 %, preferably at least, 10 %, 15 %, 20 %, 25 %,
30 %, 35 ci/o, 40 %, 45 %, or at least 50 %.
In particular, the inventors of the present application surprisingly found
that the
compounds according to the invention and mixtures thereof are effective
against a
number pathogenic microorganisms of cold and warm water fish. Alpha-pinene,
alpha-terpineol, cinnamaldehyde, dihydroeugenol, meta-cresol and terpinolene
were shown to exhibit inhibitory effect against Vibrio anguillarum, a shrimp
patho-
gen causing vibriosis.
Alpha-pinene, cinnamaldehyde, dihydroeugenol, eugenol, limonene, and meta-
cresol were shown to exhibit inhibitory effect against Aeromonas salmonicida
which is the pathogen causing a disease known as furunculosis.
is Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol,
meta-
cresol and terpinolene were shown to exhibit inhibitory effect against
Edwardsellia
tarda causing systemic infection in fish.
Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, and
terpinolene were shown to exhibit inhibitory effect against Lactococcus
garvieae
which is the etiological agent of Latococcosis, an emergent disease which
affects
many fish species and causes important economic losses both in marine and
freshwater aquaculture when water temperature increases over 16 C in summer
months.
Cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent in-
hibitory effects on the growth of Yersinia ruckeri, a pathogenic microorganism
which causes Enteric Rednnouth (ERM), a disease found especially in
salnnonids.
Cinnamaldehyde and meta-cresol were shown to exhibit inhibitory effect
against:
Vibrio salmonicida which is a psychrophilic bacterium that is the
causative agent of cold-water vibriosis in Atlantic salmon.
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(ii) Aeromonas hydrophila causing ulcers and hemorrhagic septicae-
mia. This pathogen is very resistant to conventional simple antim-
icrobials like chlorine.
(iii) Photobacterium damselae formerly Pasteurella piscicida: a pathogen
causing high losses in the culture industry of economically important
marine fishes such as seriola and red grouper in Japan and striped
bass and white perch in the United States.
(iv) Streptococcus iniae which is highly pathogenic in marine fish and is
highly lethal: outbreaks may be associated with 30-50 "1/0 mortality.
Other aquatic pathogens such as
(i) Piscirickettsia salmonis the causative agent of piscirickettsiosis or
salmonid rickettsial septicaemia (SRS),
(ii) Vibrio viscosus recently renamed MonteHa viscosa etiologically re-
sponsible for the disease referred to as "winter ulcer",
(iii) lch (parasite) one of the most prevalent protozoan parasites offish,
(iv) Vibrio harveyi, responsible for luminous vibriosis, a disease that
affects commercially-farmed prawns
will also be inhibited by the compound mixture described in the present
invention.
In a second aspect, the present invention provides a fish feed composition com-
prising at least two active compound selected from the group consisting of
alpha-
pinene (CAS 99-86-5), alpha-terpineol (CAS 98-55-5), cinnamaldehyde (CAS
14371-10-9 / 104-55-2), dihydroeugenol (CAS 2785-87-7), eugenol (CAS 97-53-
0), meta-cresol (CAS 108-39-4) and terpinolene (CAS 554-61-0). The compounds
according to the invention are commercially available or can easily be
prepared
by a skilled person using processes and methods well-known in the prior art.
As fish feed composition, the compounds of the invention can be used alone or
in
mixtures thereof, in the form of natural available extracts or extract-
mixtures or in
the form of a natural substance.
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The term "extract" as used herein includes compositions obtained by solvent ex-
traction (which are also known as "extracted oils"), steam distillation (which
are
also known as "essential oils") or other methods known to the skilled person.
Suitable extraction solvents include alcohols such as ethanol.
The term "natural" is in this context understood a substance which consists of
compounds occurring in nature and obtained from natural products or through
synthesis.
a) To the active compound(s) or natural substance or extract further
ingredients may
be added in minor amounts. Examples of such ingredients are: capsaicin,
tannin,
piperin, trimethylamine, 3,4,xylenol, furfuryl alcohol and mixtures thereof.
If a mixture of at least two compounds as specified above is preferred,
cinnamal-
is dehyde and/or meta-cresol are used as a major component of the mixture.
Suita-
bly the mixture contains 10-90 % by weight of meta-cresol and/or cinnamalde-
hyde, 1-50 % by weight of alpha-pinene, 1-50 % by weight of dihydro-eugenol,
wherein the amounts being calculated on the total amount of said components.
The total amount of these active ingredients may vary within wide limits but
is fi-
20 nally used in the fish feed from 10 to 5000 ppm, preferably between 100
and 1000
ppm, calculated on the dry weight of the fish feed.
The most preferred mixture of at least two compounds as specified above com-
prises, cinnamaldehyde and alpha-pinene as a major component of the mixture.
25 Suitably the mixture contains 30-70 % by weight of alpha-pinene, 30-70 %
by
weight cinnamaldehyde, 1-20 A by weight of metacresol, 1-20 % by weight of di-
hydro-eugenol, wherein the amounts being calculated on the total amount of
said
components. The total amount of these active ingredients may vary within wide
limits but is finally used in the fish feed from 10 to 5000 ppm, preferably
between
30 100 and 1000 ppm, calculated on the dry weight of the fish feed.
All compounds defined herein above (active compounds and additional ingredi-
ents) may be used in combination with an emulsifying surfactant. The
emulsifying
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agent can be selected advantageously from those of a rather hydrophilic
nature,
for example among polyglycerol esters of fatty acids such as esterified
ricinoleic
acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo-
glycerides, polyethylene glycol, lecithins etc.
In a preferred embodiment of the invention, a mixture of active compounds may
contain 20 % by weight of cinnamaldehyde, 20 % by weight of meta-cresol, 20 %
by weight of dihydro-eugenol, 20 % by weight of alpha-pinene, 3 % by weight of
trimethylamine, 1.8 % by weight of piperin and 4 % by weight of furfuryl
alcohol.
io In another preferred embodiment of the invention, a mixture of active
compounds
contains 40 to 60 wt.-% alpha-pinene, 40 to 60 wt.-% cinnamaldehyde, and may
further contain 1 to 5 wt.-% trimethylamine, 1 to 5 % piperin, and 3 to 8 wt.-
% fur-
furyl alcohol.
is The incorporation of the fish feed composition containing the active
compound(s)
into the fish feed may be performed as described in example 1. The mixture of
ac-
tive compounds is then prepared directly as an oil which is then mixed with
the oil
sprayed onto the feed pellets as described in example 1.
20 The incorporation of the fish feed composition containing the active
compound(s)
into the fish feed may alternatively be carried out by preparing a premix of
the ac-
tive ingredients and other suitable additives. Such a premix may comprise 2-10
%
by weight of the active mixture or natural substance or extract, 0-40 % by
weight
of other conventional additives, such as flavorings, and 50-98 % by weight of
any
25 conventional absorbing support.
The support may contain, for example, 40-50 % by weight of wood fibers, 8-10 %
by weight of stearin, 4-5 % by weight of curcuma powder, 4-5 % by weight of
rosemary powder, 22-28 % by weight of limestone, 1-3 % by weight of a gum,
30 such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 %
by
weight of water.
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This premix is then mixed with vitamins, as for example vitamin C, mineral
salts
and other feed additive ingredients, as for example yeast extracts containing
nu-
cleotides, glucan and other gut microflora modulators and then finally added
to
the feed in such quantities that the feed will contain 10-5000 ppm, preferably
100-
1000 ppm or 100-500 ppm of the active ingredients according to the invention.
Moreover, the composition of the present invention will be preferably used to-
gether with yeast extract containing nucleotides, and glucan.
Further, optional, feed-additive ingredients are coloring agents, e.g.
carotenoids
io such as beta-carotene, astaxanthin, and lutein; aroma compounds;
stabilisers;
antimicrobial peptides; polyunsaturated fatty acids; and/or at least one
enzyme
selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8);
galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC
3.4.),
phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);
is lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3);
phospholipase D
(EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); and/or
beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated
20 fatty acids, such as arachidonic acid, docosohexaenoic acid,
eicosapentaenoic
acid and gamma-linoleic acid.
The fish feed as described herein has a proximate composition of 20-60 wt.-%
protein, and 1-45 wt.-% moisture and lipid.
In some specific examples, the fish feed comprises one or more of sources of:
- protein, carbohydrate and lipid (for example, fish meal, fish oil, blood
meal,
feather meal, poultry meal, chicken meal and/or other types of meal produced
from other slaughterhouse waste),
- animal fat (for example poultry oil),
- vegetable meal (e.g. soya meal, lupin meal, pea meal, bean meal, rape
meal
and/or sunflower meal),
- vegetable oil (e.g. rapeseed oil, soya oil),
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- gluten (e.g. wheat gluten or corn gluten) and
- added amino acids (e.g. lysine)
The term "fish feed" as used herein includes a fish feed composition according
to
the invention and components as described above. Typically, fish feed includes
fish meal as a component. Suitably, fish feed is in the form of flakes or
pellets, for
example extruded pellets.
In a third aspect, the invention relates to a feed composition for aquatic
animals
and to the use of this composition for feeding fish. The feed is particularly
suitable
for feeding salmonids, including Atlantic salmon (Salmo salar), other salmon
spe-
cies and trout, and non-salmonids such as cod, sea bass, sea bream and eel.
However, it can be fed to all types offish, for example turbot, halibut,
yellow tail
and tuna.
The invention described and claimed herein is not to be limited in scope by
the
specific embodiments herein disclosed, since these embodiments are intended as
illustrations of several aspects of the invention. Any equivalent embodiments
are
intended to be within the scope of this invention. Indeed, various
modifications of
the invention in addition to those shown and described herein will become
appar-
ent to those skilled in the art from the foregoing description. Such
modifications
are also intended to fall within the scope of the appended claims.
Example 1: Preparation of Pressed Fish Feed
The main raw materials are ground and mixed. Microingredients are then added
to
the mixer and the homogenous mix is conditioned by adding water and steam to
the mass in a preconditioner. This starts a cooking process in the starch
fraction
(the binding component). The mass is fed into a pellet mill. The mass is
forced
through the mill's die and the strings are broken into pellets on the outside
of the
die. The moisture content is low and drying of the feed is not necessary.
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Additional oil including a fish feed composition according to the present
invention
is then sprayed onto the surface of pellets, but as the pellets are rather
compact,
the total lipid content rarely exceeds 24 %. The added oil may be fish oil or
vegetable oils, for example rape seed oil or soy oil, or a mixture of
vegetable oils
or a mixture of fish oil and vegetable oils. After oil coating, the pellets
are cooled
in a cooler and bagged. The final pressed fish feed contains 10 to 5000 ppm of
the composition as described in the invention.
Example 2: Method for Preparation of Extruded Fish Feed
The main raw materials are ground and mixed. Micro ingredients incl. a fish
feed
composition according to the invention are added to the mixer. The homogenous
mix is conditioned by adding water and steam to the mass in a preconditioner.
Additional oil may also be added to the mass at this stage. This starts a
cooking
process in the starch fraction (the binding component). The mass is fed into
an
extruder. The extruder may be of the single screw or the twin-screw type. Due
to
the rotational movement of the mass in the extruder, the mass is further
mixed.
Additional oil, water and steam may be added to the mass in the extruder. At
the
end of the extruder, the mass has a temperature above 100 C and a pressure
above ambient pressure. The mass is forced through the openings in the
extruder's die plate. Due to the relief in temperature and pressure, some of
the
moisture will evaporate immediately (flash off) and the extruded mass becomes
porous. The strings are cut into pellets by a rotating knife. The water
content is
rather high (18-28 %) and the pellets are therefore immediately dried to
approximately 10 % water content in a dryer.
After the dryer, more oil may be added to the feed by spraying oil onto the
surface
of the feed, or by dipping the feed in oil. It is advantageous to add the oil
to the
feed in a closed vessel where the air pressure is below ambient (vacuum
coating)
so that the porous feed pellets absorb more oil. Feed containing more than 40
%
lipid may be produced this way. After the coater, the feed is cooled and
bagged.
Oil may be added at several places in the process as explained above, and may
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be fish oil or vegetable oils, by example rape seed oil or soy oil, or a
mixture of
vegetable oils or a mixture of fish oil and vegetable oils.
Fish need protein, fat, minerals and vitamins in order to grow and to be in
good
health. The diet of carnivorous fish is particularly important. Originally in
the
farming of carnivorous fish, whole fish or ground fish were used to meet the
nutritional requirements of the farmed fish. Ground fish mixed with dry raw
materials of various kinds, such as fish meal and starch, was termed soft or
semi-
moist feed. As farming became industrialized, soft or semi-moist feed was
replaced by pressed dry feed. This was itself gradually replaced by extruded
dry
feed.
Today, extruded feed is nearly universal in the farming of a number of fish
species
such as various types of salmonid, cod, sea bass and sea bream.
The dominant protein source in dry feed for fish has been fish meal of
different
qualities. Other animal protein sources are also used for dry fish feed. Thus,
it is
known to use blood meal, bone meal, feather meal and other types of meal
produced from other slaughterhouse waste, for example chicken meal. These are
typically cheaper than fish meal and fish oil. However, in some geographic
regions, there has been a prohibition against using such raw materials in the
production of feeds for food-producing animals and fish.
It is also known to use vegetable protein such as wheat gluten, maize (corn)
gluten, soya protein, lupin meal, pea meal, bean meal, rape meal, sunflower
meal
and rice flour.
Example 3: Evaluation of the effect of the active compounds according to
the invention on survival of Yersinia ruckeri, Vibrio anguillarum, Vibrio sal-
monicida, Aeromonas salmonicida, Aeromonas hydrophila, Edwardsellia
tarda, Photobacterium damselae, Lactococcus garvieae, Streptococcus
iniae.
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The antimicrobial activity of the composition of the invention towards
Yersinia
ruckeri, Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida, Aeromo-
nas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus gar-
vieae, and Streptococcus iniae were determined in vitro.
In the tests the following organisms, growth media, culture conditions and
evalua-
tion method were used:
Bacteria: All tested pathogenic strains belong to the strain collection of the
Cen-
tre for fish and wildlife health, Institute of Animal Pathology, University of
Bern
(Switzerland).
Determination of suitable bacteria dilution: From a 24 hour old subculture of
bacteria on blood sheep agar (Biomerieux, Geneva) a small amount was trans-
ferred to sterile NaCI until a McFarland value of 0.5 was obtained. From this
solu-
tion 3, 1.5 and 0.75 % dilutions in TSB were made on 96 well plates with round
bottoms. Each well received a total volume of 100 pl. After 24 hours at 22 C
the
growth of bacteria was assessed. The dilution resulting in a well demarcated
spot
covering half of the round bottom well was selected for the experiments.
Determination of solvent effect: To solve test substances in TSB agar, alcohol
(ETOH) was used. To determine a possible effect of alcohol, the calculated
final
concentrations of alcohol in the test wells (0.1, 0.05 and 0.025 %) were
tested
with different concentrations of bacteria, and did not show any effect on
bacterial
growth.
Concentration of test substances: The in vitro dose range was estimated con-
sidering a probable dietary concentration of at least 1000 ppm and a daily
feeding
rate of 2 %. The potential concentration in the gut was established at maximum
0.1 p1/100 pl. A serial 2 dilution was then tested leading to final
concentrations of
the substance of 0.85 pg/nril; 0.42 pg/nril and 0.21 pg/nril when adjusted to
average
essential oil density.
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From each substance a stock solution consisting of 2 pl substance, 18 pl ETOH
and 180 pl PBS was prepared.
Preparation of Plates: Triplicates of three concentrations (0.21, 0.42 and
0.85
pg/nril) of each substance were tested. On each plate a positive control
consisting
of bacteria in TSB and a blank control (PBS) was included. Further Triplicates
of
three dilutions of ETOH were also included on each plate.
Reading of plates: After an incubation of 24 hours at 22 C the plates were
read
using a score of 0 (no bacterial growth = no dot on the bottom of the well) to
3
(normal growth = size of dot comparable to dot of positive control).
The following results were obtained and are summarized in Table 1:
Suitable concentration of bacteria for test: A concentration of 1.5 % of 0.5
McFarland value resulted in a clearly visible spot covering 2/3 of a round
bottom
well. This concentration was therefore considered as suitable for the tests.
Effect of ETOH on bacterial growth: No effect of any of the applied alcohol
con-
centrations (1 %, 0.5 % and 0.05 %) could be found.
Effect of tested substances: The results are summarized in Table 1. No major
differences within triplicates nor between triplicates on different plates
could be
seen. All positive controls showed clearly visible growth of bacteria within
48
hours. As expected from the preliminary trials no negative effect of alcohol
could
be determined. Broadest spectrum effects on the bacteria were found with cinna-
maldehyde, and alpha-pinene followed by meta-cresol and dihydro-eugenol.
Table 1: Effect of different substances on growth of Yersinia ruckeri, Vibrio
anguil-
larum, Vibrio salmonicida, Aeromonas salmonicida, Aeromonas hydrophila, Ed-
wardseffia tarda, Photobacterium damselae, Lactococcus garvieae, and Strepto-
coccus iniae after 48 hours incubation at 22 C. Values represent means of 5
indi-
vidual triplicates. nd: not determined, 3: no effect, 0: maximum growth
inhibition.
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Table 1:
L:1)
Rs 0
Rs cis cD
CD 0 '0 10 2 (13 to .92
"Z.3 -a E
E=- =-
: c c -a c
0 0
>: E E g 14i oi
alpha-pinene 1 nd 0 nd 0 nd 0 nd 0.22
nd
alpha-terpineol 3 nd 0 nd 1.44 nd 0 nd 0.33
nd
cinnamaldehyde0 0.02 0 0.02 0 0 0 0 0 0
dihydro-eugenol 0 nd 0 nd 0 nd 0 nd 0 nd
Lc) eugenol 0 nd 0.56 nd 0 nd 0 nd 0.56
nd
co
limonene 3 nd 1 nd
0 nd 0 nd 1 nd
meta-cresol 0 0.02 0 0.02 0 0 0 0 1 0
terpinolene 3 nd 0 nd 1.44 nd 0 nd 0.33
nd
alpha-pinene 3 nd 0 nd 0 nd 0 nd 2.67
nd
alpha-terpineol 3 nd 0 nd 3 nd 0 nd 2.67
nd
cinnamaldehyde0.27 2.33 0 0.02 0 0 0 0 2.93 0.27
15). dihydro-eugeno13 nd 1 nd 1.33 nd 2.6 nd 1 nd
CNI eugenol 2.89 nd 1.67 nd 0.78 nd 1.9 nd
1.67 nd
limonene 3 nd 2.67
nd 0.67 nd 0.1 nd 2.67 nd
meta-cresol 2.13 0.73 0.6 0.02 0 0 0 0 3 1.13
terpinolene 3 nd 0 nd 3 nd 0 nd 2.67
nd
alpha-pinene 3 nd 0 nd 0 nd 0 nd 3 nd
alpha-terpineol 3 nd 3 nd 3 nd 0.11 nd 3 nd
cinnamaldehyde2.8 3 0.2 0.02 0.73 0 0.47 0 3
3
dihydro-eugenol 3 nd 2 nd 2.56 nd 3 nd 2 nd
eugenol 3 nd 3 nd
2 nd 3 nd 3 nd
limonene 3 nd 3 nd 2 nd 1.44 nd 3 nd
meta-cresol 3 1 1.53 0.2 0.13 0 1.93 1.4 3
3
terpinolene 3 nd 3 nd 3 nd 0.11 nd 3 nd
Example 4: Evaluation of the effect of the active compounds according to
the invention on survival of Vibrio anguillarum, and Aeromonas salmonicida.
The effect of combinations of different substances was tested for growth
inhibition
of Aeromonas salmonicida and Vibrio anguillarum after 48 hours of incubation
at
22 C. The conditions of the tests were as described in example 3 and
concentra-
tions tested were (0.21 pg/ml, 0.42pg/m1 and 0.85pg/m1).Values of Table 2 and
3
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represent the mean of 3 individual triplicates on three different plates for
Aeromo-
nas salmonicida and Vibrio anguillarum, respectively. 3: means no effect, 0:
means maximum growth inhibition.
The substances tested either alone or in combinations are meta-cresol,
cinnamal-
dehyde, alpha-pinene, dihydroeugenol. Mixtures of the substances were prepared
in proportions 1:1 for two substances, 1:1:1 for three substances and 1:1:1:1
for
the four substances.
Results: All compounds and combinations of compounds have shown some in-
hibitory effect against the two pathogens tested at the highest concentration,
and
a clear dose dependence of the effects against the two bacterium species was
observed and is shown in Table 2 and Table 3.
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Table 2: Results of inhibition assay of Aeromonas salmonicida
Product Concentrations
0.85 pg/ml 0.42 pg/ml 0.21 pg/ml
meta-cresol 0.02 0.02 0.33
cinnamaldehyde 0.02 0.02 0.33
alpha-pinene 0.02 0.22 2
dihydroeugenol 0.02 1.33 3
meta-cresol / cinnamaldehyde 1:1 0.02 0.22 2
meta-cresol / alpha-pinene 1:1 0.02 0.22 1.67
meta-cresol / dihydroeugenol 1:1 0.02 1.11 2.33
cinnamaldehyde / alpha-pinene 1:1 0.02 0.11 1.78
cinnamaldehyde/dihydroeugenol 1:1 0.02 0.89 3
alpha-pinene / dihydroeugenol 1:1 0.02 1.78 3
meta-cresol / cinnamaldehyde / alpha-pinene
0.02 0.33 2
1:1:1
meta-cresol / cinnamaldehyde / dihydroeugenol
0.67 2
1:1:1 0.02
meta-cresol / alpha-pinene / dihydroeugenol
0.02 0.89 2.33
1:1:1
cinnamaldehyde / alpha-pinene / dihydroeugenol
0.02 1.22 2.44
1:1:1
meta-cresol / cinnamaldehyde / alpha-pinene /
0.02 1.33 2.33
dihydroeugenol 1:1:1:1
ETOH 3 3 3
Positive control 3
Negative control 0.02
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Table 3: Inhibition of Vibrio anguillarum
Product Concentrations
0.85 pg/ml 0.42 pg/ml 0.21 pg/ml
meta-cresol 0.02 2.67 3
cinnamaldehyde 0.02 0.02 1
alpha-pinene 0.02 0.02 0.02
dihydroeugenol 0.02 1.56 2.67
meta-cresol / cinnamaldehyde 1:1 0.02 0.89 2.67
meta-cresol / alpha-pinene 1:1 0.02 0.02 2.67
meta-cresol / dihydroeugenol 1:1 0.02 2.22 3
cinnamaldehyde / alpha-pinene 1:1 0.02 0.02 0.02
cinnamaldehyde / dihydroeugenol 1:1 0.02 0.11 2.89
alpha-pinene / dihydroeugenol 1:1 0.02 0.02 1.78
meta-cresol / cinnamaldehyde / alpha-pinene
0.02 0.02 2.78
1:1:1
meta-cresol / cinnamaldehyde / dihydroeugenol
2.33 3
1:1:1 0.02
meta-cresol / alpha-pinene / dihydroeugenol
0.02 2.33 2.89
1:1:1
cinnamaldehyde / alpha-pinene / dihydroeugenol
0.02 0.67 3
1:1:1
meta-cresol / cinnamaldehyde / alpha-pinene /
0.02 2.22 3
dihydroeugenol 1:1:1:1
ETOH 3 3 3
Positive control 3
Negative control 0.02
Example 5: Effect of a combination of essential oils comprising alpha-
s pinene, cinnamaldehyde, dihydroeugenol and meta-cresol on the mortality
of rainbow trout challenged with Aeromonas salmonicida.
A challenge experiment with Aeromonas salmonicida has been set up with juve-
nile rainbow trout in order to test the effect of three doses of a combination
of four
essential oils supplemented to the feed.
Fish: 600 young of the year rainbow trout were obtained from a commercial fish
farm in Switzerland.
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Fish rearing conditions: For the acclimatization period and the experimental
feeding period before infection, fish from the same dietary treatment are kept
in
130 l glass aquaria equipped with a tap water flow through system and constant
aeration. Water temperature is maintained at 17 1.0 'C.
At the time of infection, fish from each treatment are transferred into four
38 l
glass aquaria equipped with a flow through system with tap water and constant
aeration. Water temperature is maintained at 17.5 1.0 C.
Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4
mm diameter. From these pellets, crumbles adapted to the size of the fish were
produced and ca 5-kg batch of feed were produced by coating the essential oil
combination at the different doses mixed with the oil onto the crumbles.
Coating
was performed using a peristaltic pump and a concrete mixer to insure homoge-
nous distribution of the products.
Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was
regularly
adjusted to the weight gain offish.
The four experimental diets were produced:
- Diet A: a control diet not supplemented with essential oil combination
- Diet B: basal diet supplemented with 500 ppm of the essential oil
combination
- Diet C basal diet supplemented with 2000 ppm of the essential oil
combination
- Diet D: basal diet supplemented with 4000 ppm of the essential oil
combination
The essential oil combination was the following one: al pha-pinene, cinnamalde-
hyde, dihydroeugenol and meta-cresol at equivalent doses. The experimental di-
ets were fed for four weeks prior to challenge and after challenge until the
end of
the experiment. Fish weight was recorded at the start of experimental feeding,
be-
fore challenge and at the end of the experiment on the remaining fish.
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Challenge experiment: The challenge dose was determined in a preliminary ex-
periment, on the same fish population and under the same conditions. After a
pe-
riod of 4 weeks of experimental feeding, 25 fish were randomly distributed
into
four 38 l glass aquaria. Fish from each experimental tank were then
anaesthetized
and challenged by i.p. injection of Aeromonas salmonicida at a dose of 1.2*102
cfu/fish determined by spectrophotometry. The daily mortality was then
recorded
over a period of three weeks. First dead fish from each glass aquarium were
sub-
jected to a bacteriological investigation to confirm the bacteriological
aetiology
causing death. At the end of the experiment, all surviving fish were assessed
for
external signs of infection and in addition from 6 fish per diet group a
necropsy
and bacteriology were performed.
Results of cumulative mortality are presented in Table 4:
Table 4: Mean + SD of cumulative mortality in relation to essential oil combi-
nation dose
Day after
challenge Control 500 ppm 2000 ppm 4000 ppm
Mean Stdev Mean Stdev Mean Stdev Mean Stdev
0 0 0 0 0 0 0 0 0
1 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0
4 7.0 2.0 8.0 7.3 2.0 2.3 0.0 0.0
5 14.0 7.7 15.1 6.8 10.0 9.5 6.0 6.9
6 16.0 8.6 22.2 11.5 11.0 10.5 7.0
6.8
7 16.0 8.6 23.3 11.5 11.0 10.5 8.0
6.5
8 17.0 8.9 23.3 11.5 12.0 11.8 9.0
8.2
9 20.0 11.3 23.3 11.5 12.0 11.8 10.0
10.1
10 20.0 11.3 23.3 11.5 12.0 11.8 10.0
10.1
11 20.0 11.3 23.3 11.5 12.0 11.8 12.0
13.9
12 20.0 11.3 23.3 11.5 12.0 11.8 12.0
13.9
13 21.0 12.4 23.3 11.5 12.0 11.8 14.0
17.7
14 21.0 12.4 23.3 11.5 12.0 11.8 14.0
17.7
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15 21.0 12.4 23.3 11.5 12.0 11.8 15.0
19.7
16 21.0 12.4 23.3 11.5 12.0 11.8 15.0
19.7
17 21.0 12.4 23.3 11.5 12.0 11.8 15.0
19.7
18 21.0 12.4 23.3 11.5 12.0 11.8 15.0
19.7
19 21.0 12.4 23.3 11.5 12.0 11.8 15.0
19.7
Results: Feed intake was normal and fish grew from 4 to ca 14 g during the ex-
periment.
The results reveal a dose dependent effect of essential oils in rainbow trout
chal-
lenged with A. salmonicida. Lowest mean values were found in the two groups re-
ceiving the higher doses, while in the group receiving the lowest dose a
slightly
higher cumulative mortality than in the control was found. These results
suggest
that the mixture of 4 essential oils in the diet have a beneficial effect on
rainbow
trout infected with A. salmonicida. Differences were found in cumulative
mortality
and in the presence of bacteria in surviving fish at the end of the
experiment.
Example 6: Effect of an essential oil combination comprising alpha-pinene
and cinnamaldehyde on the mortality of rainbow trout challenged with
is Aeromonas salmonicida.
Aim: A challenge experiment with Aeromonas salmonicida has been set up with
juvenile rainbow trout in order to test the effect of alpha-pinene and
cinnamalde-
hyde supplemented to the feed.
Fish: 600 young of the year rainbow trout were obtained from DSM Nutritional
Products
Fish rearing conditions: For the acclimatization period and the experimental
feeding period before infection, fish from the same dietary treatment are kept
in
130 l glass aquaria equipped with a tap water flow through system and constant
aeration. Water temperature is maintained at 14.5 1.0 C.
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At the time of infection, fish from each treatment are transferred into four
38 l
glass aquaria equipped with a flow through system with tap water and constant
aeration. Water temperature is maintained at 13.5 1.0 C.
Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4
mm diameter. From these pellets, crumbles adapted to the size of the fish were
produced and ca 5-kg batch of feed were produced by coating the essential oil
combination at the different doses mixed with the oil onto the crumbles.
Coating
was performed using a peristaltic pump and a concrete mixer to insure homoge-
nous distribution of the products.
Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was
regularly
adjusted to the weight gain offish.
Three experimental diets were produced:
- Diet A: a control diet not supplemented with essential oil combination
- Diet B: basal diet supplemented with 4000 ppm of alpha-pinene
- Diet C: basal diet supplemented with 4000 ppm of alpha-pinene +
cinnamalde
hyde at equal doses
The experimental diets were fed for four weeks prior to challenge and after
chal-
lenge until the end of the experiment.
Fish weight was recorded at the start of experimental feeding, before
challenge
and at the end of the experiment on the remaining fish.
Challenge experiment: The challenge dose was determined in a preliminary ex-
periment, on the same fish population and under the same conditions. After a
pe-
riod of 4 weeks of experimental feeding, 25 fish were randomly distributed
into
four 38 l glass aquaria. Fish from each experimental tank were then
anaesthetized
and challenged by i.p. injection of Aeromonas salmonicida at a dose of 5'103
cfu/fish determined by spectrophotometry. The daily mortality was then
recorded
over a period of three weeks. First dead fish from each glass aquarium were
sub-
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jected to a bacteriological investigation to confirm the bacteriological
aetiology
causing death. At the end of the experiment, all surviving fish were assessed
for
external signs of infection and in addition from 6 fish per diet group a
necropsy
and bacteriology were performed.
Results: Feed intake was normal and fish grew from 1 to ca 8 g during the ex-
periment.
The results reveal no effect of alpha-pinene alone but there is a positive
effect of
the combination of alpha-pinene + cinnannaldehyde in reducing mortality by 15
%
against the control treatment. Results of cumulative mortality are presented
in
Table 5:
Table 5: Mean + SD of cumulative mortality in relation to essential oil combi-
nation
Alpha-pinene + cin-
Day after Alpha-pinene
Control namaldehyde (2000
challenge (4000 ppm)
ppm each)
Mean Stdev Mean Stdev Mean Stdev
0 0 0 0 0 0 0
1 0 0 0 0 0 0
2 0 0 0 0 0 0
3 0 0 0 0 0 0
4 0 0 0 0 0 0
5 0 0 0 0 1 2
6 4.1 5.9 5.0 3.8 2.0 2.3
7 16.2 9.9 21.1 14.3 11.0 3.8
8 25.3 8.6 37.3 11.2 22.0 5.2
9 28.3 10.0 40.3 9.5 24.0 5.7
10 30.4 12.3 41.4 8.6 25.0 6.8
11 33.4 13.5 43.4 9.1 27.0 3.8
12 33.4 13.5 43.4 9.1 27.0 3.8
13 34.4 12.7 43.4 9.1 28.0 3.3
14 34.4 12.7 43.4 9.1 29.0 3.8
